AMP-activated protein kinase (AMPK) acts as a cellular low fuel warning system. Increases in AMP concentration ([AMP]) activate AMPK, while high concentrations of ATP ([ATP]) antagonize AMPK activation. After this warning system is activated it initiates protective measures to either conserve ATP or promote alternative methods of ATP generation. We increased [AMP] using metabolic inhibitors and measured the relationship between AMPK activity and [AMP] in the perfused rat heart. AMPK's half maximal activating concentration of AMP was 1.8 +/- 0.3 muM and 10 to 30 muM cytosolic [AMP] resulted in a two-fold maximal increase in AMPK activity. In this study [ATP] was greater than or equal too 7 mM. These results suggest that in the heart ATP does not antagonize AMP activation as it does in test tube studies. In the ischemic rat heart we found that 10 to 30 fM cytosolic [AMP] resulted in an eight-fold increase in AMPK activity. This four times greater AMPK activation in ischemia indicates that factors other than AMP cause AMPK activation. These observations lead to our first hypothesis that the causal factors for AMPK activation are the metabolically active cytosolic [AMP] (not total AMP), reduced [ATP], and intracellular H+ ([H+]i). We propose two specific aims to test this hypothesis. First, to define the relationship between AMPK activity and the [AMP] in the isolated perfused mouse heart. The [AMP], [ATP], and [H+]i, will be altered using three manipulations of energy metabolism, metabolic inhibitors, hypoxia, and ischemia. Second, to define how long AMPK remains activated after restoration of normal [AMP] in the isolated perfused mouse heart. Our second hypothesis that the predominant consequence of AMPK activation is a shift in substrate metabolism, in particular, an acceleration of fatty acid oxidation resulting from acetyl-CoA carboxylase phosporylation by AMPK. The third specific aim will define the alteration in substrate utilization resulting from increased AMPK activity in the isolated perfused mouse heart. Our long-term objective is to determine the causes of AMPK activity and the consequences of AMPK activity for myocardial substrate metabolism during the two most dangerous cardiovascular diseases, ischemia and heart failure, in the United States.